206 CHAPTER XVI. 



0'75 per cent, salt solution, and dilutes it with 15 to 20 

 volumes of sea-water. Seidbnmann {Zeit. wiss. Mik., xvii, 

 1900, p. 239) takes for tlie choroid a solution of 0"02 per cent, 

 in 0"5 per cent, salt solution. Lavdowsky {ibid., xii, 1895, 

 p. 177) takes ^V ^'^ k P^r cent, in white of egg, or serum. 

 Similarly YouNG {ibicL, xv, 1898, p. 253). Michailow (ibid., 

 xxvii, 1910, p. 10) takes ^ to ^'^ per cent, in Ringei''s salt 

 solution (for nerves of Mammals). 



Apathy {Zeit. wiss. Mil-., ix, 1892, p. 15 ; see also his 

 Mihrotechnih , p. 172) proceeds as follows for Hirudinea and 

 other invertebrates. A portion of the ventral cord is ex- 

 posed, or dissected out. If it be desired to stain as many 

 ganglion cells as possible, as well as fibres, the lateral nerves, 

 as well as the connectives, should be cut through near a 

 ganglion. The preparation is then treated with the stain. 

 This is, for the demonstration chiefly of fibres in Hirudo and 

 Fontobdella, either a 1 : 1000 solution in 0'5 to 0'75 per cent, 

 salt solution, allowed to act for ten minutes ; or a 1 : 10,000 

 solution allowed to act for an hour to an hour and a half ; or 

 a 1 : 100,000 solution allowed to act for three hours {Liim- 

 bricus requires twice these times; Astacus and Unio require 

 three times ; medullated nerves of vertebrates four times) . 

 For the demonstration of ganglion cells the stain is allowed 

 to act three or four times as long. 



The preparations from the 1 : 1000 solution are then 

 washed in salt solution for an hour ; those from the 1 : 10,000 

 solution for a quarter of an hour ; those from the 1 : 100,000 

 solution need not be washed at all. They are then treated 

 with one of the ammoniacal fixing and differentiating liquids 

 described in § 343. This is done by pouring the liquid over 

 them, and leaving them in it without moving them about in it 

 for at least an hour, and by preference in the dark. The 

 further treatment is as described in § 343. 



The object of the ammonia in these liquids is to differ- 

 entiate the stain — to produce an artificial " secondary diffe- 

 rentiation." It acts by washing out the absorbed colour 

 from certain elements, others resisting longer. 



See also, for Hirudinea, Sanchez, in Trab. Lab. Invest. 

 Biol. Univ. Madrid, vii, 1909, fasc. 1-4, or Zeit. wiss. Mik., 

 xxvii, 1910, p. 393 (injection of solutions of 0'2, O'l, or 0"05 

 per cent,, with further treatment as Apathy or Bethe). 



